We plan to continue our studies of the different NACMs from the pathogenic and non-pathogenic amebae. By necessity, when these were found, they had been isolated under preparative conditions, that is, under conditions from which we could reclaim the fractionated material in a manner that did not interfere with the detection of the cytopathogenic activity as measured in tissue cultured cells. Now that we have the means available to produce small quantities of the relatively "clean" NACMs, we plan to use them for analytical measures including that of homogeneity and molecular weight by SDS polyacrylamide gel electrophoresis, and charge by analytical isoelectric focusing. Problems in the purification will be explored more fully, such as that of separating NACM from the gel or reducing inactivation during isoelectric focusing to obtain better yields of material as well as the use of other materials for isolations, such as ion-exchange resins and gels. The purified NACMs will be studied spectrophotometrically, enzymatically by assays before and after digestion, and biologically by inoculation into tissue cultured cells and into mice. Quantitative determinations of the NACM yield per ameba for the different ameba strains related to the ratios of the large and small NACMs for the strain may be very significant in relating the molecular structures to the pathogenic activity of the amebae in mice, and we hope to be able to begin these studies. When sufficient quantities of the different purified NACMs are available, they will be used to inoculate hamsters for the production of antibodies for immunologic investigations.